title: Substrate-ligand interactions in Geobacillus stearothermophilus nitric oxide synthase.
abstract: Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [l-arginine (l-Arg) --> N-hydroxy-l-arginine (NOHA) --> l-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence, it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, we found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of nu(Fe-CO)/nu(C-O) modes in the resonance Raman spectra. In the nu(Fe-CO) versus nu(C-O) inverse correlation plot, one set of data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of l-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential into the distal heme pocket. To assess how substrate binding affects Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm(-1), similar to that of bsNOS. The binding of l-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features with respect to the oxygen chemistry of NOS is discussed.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Mariam
authors.initials: M
authors.lastname: Kabir
authors.forename: Jawahar
authors.initials: J
authors.lastname: Sudhamsu
authors.forename: Brian R
authors.initials: BR
authors.lastname: Crane
authors.forename: Syun-Ru
authors.initials: SR
authors.lastname: Yeh
authors.forename: Denis L
authors.initials: DL
authors.lastname: Rousseau
mesh_terms: D000042:Absorption; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001406:Bacillaceae / Q000201:enzymology*; D001708:Biopterins / Q000737:chemistry / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008024:Ligands; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D013059:Spectrum Analysis, Raman; D013455:Sulfur / Q000378:metabolism
chemical_list: D003067:Coenzymes; D008024:Ligands; D001708:Biopterins; D013455:Sulfur; D002248:Carbon Monoxide; D001120:Arginine; D007501:Iron; D019001:Nitric Oxide Synthase; D010100:Oxygen